The invention relates to novel microorganisms of the genus Actinomadura, Amycolatopsis or Rhodococcus, and to a novel process for the preparation of amides using these microorganisms or using enzyme extracts of these microorganisms.
For amides such as, for example, nicotinamide, a vitamin of the vitamin B complex which is essential to animals and man, a number of biotechnological processes are already known. Generally, it is known that microorganisms containing nitrile hydratase convert nitriles to the corresponding amides. Thus EP-A-0 188 316 describes a process for the preparation of nicotinamide starting from 3-cyanopyridine using microorganisms of the genus Rhodococcus, Arthrobacter or Microbacterium.
A disadvantage of this process is that these microorganisms have only a low activity for the conversion of 3-cyanopyridine to nicotinamide.
EP-A-0 307 926 describes the conversion of 3-cyanopyridine to nicotinamide by means of microorganisms of the species Rhodococcus rhodochrous J1. In order that these microorganisms catalyse the desired conversion, they must be induced.
A further disadvantage of this process is that Rhodococcus rhodochrous J1 is red-coloured and accordingly a discoloration of the product takes place. In addition, this microorganism has a low heat stability and is inhibited, for example, by the substrate 3-cyanopyridine.
A further process for the preparation of nicotinamide starting from 3-cyanopyridine by means of microorganisms of the species Rhodococcus rhodochrous J1 is described in EP-A-0 362 829. In order to increase the specific activity of the microorganisms containing nitrile hydratase, urea or a urea derivative was added to the culturing medium as an inducer. As in the process described beforehand, a discoloration of the product also takes place in this process.
In addition, WO 95/17 505 describes a process for the preparation of aromatic amides starting from the corresponding nitriles by means of microorganisms of the species Rhodococcus rhodochrous M33. A disadvantage of this process is the red colouration of Rhodococcus rhodochrous M33 and also the high KM value for the substrate 3-cyanopyridine.
The object of the present invention was to eliminate these disadvantages and to make available a process for the preparation of amides in which the corresponding amides can be isolated in good yield and purity.
This object is achieved by the novel microorganisms according to claims 1 and 3, and by the process according to claim 6.
According to the invention, the process is carried out by converting a nitrile, as substrate, to the corresponding amide by means of microorganisms of the genus Actinomadura, Amycolatopsis or Rhodococcus, using an enzyme extract of these microorganisms or by means of purified nitrile hydratase of microorganisms of the genus Amycolatopsis or Actinomadura.
The nitriles employed for the biotransformation such as, for example, 3-cyanopyridine are commercially available compounds.
The microorganisms according to the invention are able to convert nitriles as substrates into the corresponding amides. Preferably, these microorganisms have the ability to grow on nitriles or amides as the sole C and/or N source.
The microorganisms according to the invention are obtainable by means of suitable selection, for example, from soil samples, sludge or waste water with the aid of customary microbiological techniques. Expediently, the microorganisms are selected by growth with nitriles or amides as the preferably sole C and N source in the presence of cobalt ions. Nitriles and amides suitable for selection are, in particular, the nitriles also employed as substrates in the later biotransformation and the corresponding amides obtainable therefrom. Suitable growth media are likewise known to the person skilled in the art, for example the medium described in Table 1 can be used.
Customarily, the microorganisms are cultured (grown) in the same manner even before the actual biotransformation, the abovementioned media being used.
As known professionally, a nitrile hydratase is only formed when the growth medium contains cobalt ions as a cofactor. Suitable xe2x80x9ccobalt compounds generating cobalt ionsxe2x80x9d are Co2+ or Co3+ salts. Examples of Co2+ and Co3+ salts are cobalt chlorides, cobalt sulphates and cobalt acetates.
Expediently, the cobalt compound employed is a Co2+ salt such as, for example, CoCl2. Growth, however, can also be carried out in the presence of vitamin B12 together with metallic cobalt or other cobalt compounds which generate a cobalt ion in situ. Expediently, the cobalt compound is employed in an amount from 1 to 10 mg/l, preferably from 1 to 3 mg/l.
Customarily, growth is carried out at a temperature from 20 to 50xc2x0 C. and at a pH between pH 5 and pH 8, preferably from 30 to 45xc2x0 C. and between pH 5.5 and pH 7.5.
The actual biotransformation can be carried out using microorganisms of the genus Actinomadura, Amycolatopsis, using an enzyme extract of these microorganisms or by means of purified nitrile hydratase from these microorganisms. Expediently, the biotransformation is carried out using microorganisms of the species Actinomadura spadix, for example the isolates Actinomadura spadix E3733, Actinomadura spadix E3736, Actinomadura spadix 45A32, Actinomadura spadix 4501 or Actinomadura spadix C15. The biotransformation is preferably carried out using microorganisms corresponding to the species Amycolatopsis NE 31 and Amycolatopsis NA40 or their functionally equivalent variants and mutants. Microorganisms corresponding to the species Amycolatopsis NA40 are particularly preferably employed. Microorganisms of the species mentioned were deposited on 03.06.1997 in the Deutschen Sammlung von Mikroorganismen und Zellkulturen GmbH [German Collection of Microorganisms and Cell Cultures GmbH], Mascheroderweg 1b, D-38124 Brunswick under the designations Amycolatopsis NE 31 and Amycolatopsis NA40 according to the Budapest Convention and have the deposit numbers DSMZ 11616 and DSMZ 11617 respectively. These two microorganisms have been more accurately identified and are to be assigned to species of the genus Amycolatopsis which have not yet been described in the literature.
Accordingly, the invention also relates to microorganisms of the genus Amycolatopsis or Actinomadura which are capable of converting an amide into a nitrile, in particular microorganisms with the designation Amycolatopsis NA40 (DSMZ 11617) and Amycolatopsis NE31 (DSMZ 11616).
In addition, it has been found that specific microorganisms of the genus Rhodococcus have better properties for the conversion of nitriles to amides than the Rhodococcus rhodochrous J1 described in EP-A-0 362 829. These microorganisms are Rhodococcus GF674, Rhodococcus GF578, Rhodococcus GF473, Rhodococcus GF270 (DSMZ 12211) and Rhodococcus GF376 (DSMZ 12175) or their functionally equivalent variants and mutants. The microorganism DSMZ 12175 was deposited on 15.5.1998 and the microorganism DSMZ 12211 on 8.6.1998 in the Deutschen Sammlung von Mikroorganismen und Zellkulturen GmbH [German Collection of Microorganisms and Cell Cultures GmbH] according to the Budapest Convention.
The Rhodococcus strains GF270, GF376, GF473, GF578 and GF674 have been assigned according to identification to species of the genus Rhodococcus which are not yet described in the literature. Accordingly, the invention also relates to the microorganisms Rhodococcus GF270, Rhodococcus GF376, Rhodococcus GF473, Rhodococcus GF578 and Rhodococcus GF674.
Unlike the microorganisms of the genus Actinomadura or Amycolatopsis, the microorganisms of the genus Rhodococcus are expediently induced before the actual conversion. Suitable inducers are those described in EP-A-0 307 926, such as, for example, acetamide, butyramide, methacrylamide, propionamide, crotonamide and valeramide.
xe2x80x9cFunctionally equivalent variants and mutantsxe2x80x9d is understood as meaning microorganisms which are derived from the abovementioned source organisms and essentially have the same characteristics and functions as these. Variants and mutants of this type can be formed by chance, e.g. by UV irradiation or by mutagenic chemicals.
Abbreviations and explanations for the identification
Sugar types according to Lechevalier et al. 1971 Fatty acid types according to Kroppenstedt 1985 and 1992.
Identification of GF270, GF376, GF473, GF578 and GF674
The identification of these strains is based on 5 characteristics which are independent of one another.
1. Morphology and colour of the colonies: short-branched hyphae, which disintegrate into rod- and spore-like elements. The colonies of GF270 and GF376 are salmon-pink (RAL 3022) and those of GF578 and GF674 are light red (RAL 3012).
2. Diamino acids of the peptidoglycan: meso-diamino-pimelic acid
3. Mycolic acids: Rhodococcus mycolic acids: The determination of the long-chain mycolic acid was carried out by means of high-temperature gas chromatography. The elution profiles of the mycolic acids of GF270 and GF376 and those of GF473, GF578 and GF674 were identical. The mycolic acid length for GF270 and GF376 was C38-C46 and that for GF473, GF578 and GF674 was C40-C48. The mycolic acid patterns were compared with mycolic acid patterns of Rhodococcus strains. GF270 was identified with a very low correlation factor (0.086) as belonging to Rhodococcus rhodochrous; it was not possible to identify GF376 by this method. The other three isolates GF473, GF578 and GF674 were identified with a very low correlation factor as belonging to Rhodococcus ruber.
4. Fatty acid pattern: unbranched, saturated and unsaturated fatty acids including tuberculostearic acid. The fatty acid pattern is diagnostic of all Rhodococcus genera and closely related Mycobacterium, Nocardia, Dietzia, Tsukamurella and some Corynebacteria species. The identification at the species level was obtained by qualitative and quantitative differences in the fatty acid pattern of GF270, GF376, GF473, GF578 and GF674 with the fatty acid patterns of Rhodococcus species.
5. The 16S rDNA subsequences of GF270 and GF376 were identical (100%), although the comparison of them with the Rhodococcus strains only showed 99.1% similarity to the closest related Rhodococcus rhodochrous. GF473 and GF578 were identical in their 16S rDNA sequence (100%). GF674 differs from GF578 in only one base pair of 500 (99.8%). All three isolates show only a distant relationship with Rhodococcus coprophilus (98.4%).
Based on the chemotaxic and molecular biology results, it can be concluded that GF270 and GF376 on the one hand and GF473, GF578 and GF674 on the other hand are strains of 2 new Rhodococcus species. GF270 and GF376 are closely related to Rhodococcus rhodochrous in their 16S rDNA (99.1%), however GF473, GF578 and GF674 are only distantly related to Rhodococcus coprophilus (98.4%).
The enzyme extract can be obtained by professionally customary disruption of the microorganisms, such as, for example, by disruption by means of ultrasound, by means of the French press method or the lysozyme method. This enzyme extract and, of course, also the complete microorganism cells can be immobilized on a suitable support material, customarily embedded in a polymer, for carrying out the process, or absorbed on a suitable support material.
The enzymes according to the invention having nitrile hydratase activity are obtainable from the microorganisms of the genus Amycolatopsis and are able to convert a nitrile into an amide, in particular they are obtainable from Amycolatopsis NA40 (DSMZ 11617).
These enzymes in particular have the following properties:
a) a pH optimum of pH 6.5xc2x11.0
b) a temperature optimum between 35 and 40xc2x0 C. at a pH of 7.0
c) a KM value for the substrate 3-cyanopyridine of 41.7 mMxc2x17.7 mM (20xc2x0 C., 45 mM phosphate buffer, pH 7.0)
in particular the enzymes have a
d) molecular weight of 106 kDa, such as, for example, determined by SDS-PAGE.
Nitriles can generally be employed as substrates for the biotransformation. Expediently, either aliphatic nitriles having 1 to 10 carbon atoms, optionally substituted by, for example, hydroxyl, amino, halogen or carboxyl, or substituted or unsubstituted aromatic nitriles having 4 to 10 carbon atoms in the aromatic ring system are employed. Aliphatic nitriles having 1 to 10 carbon atoms which can be used are dinitriles, hydroxynitriles, aminonitriles such as, for example, n-octanenitrile, cyanoacetic acid, isocapronitrile, n-valeronitrile, adiponitrile, glutaronitrile, succinonitrile, sebaconitrile, propionitrile, crotononitrile, acrylonitrile, methacrylonitrile, n-butyronitrile or azelanitrile. Aromatic nitriles having 4 to 10 carbon atoms which can be used are nitriles of the general formula 
in which R1 and R2 are a hydrogen atom, a halogen atom or C1-4-alkyl. F, Cl, Br or I can be used as halogen atom. Methyl, ethyl, propyl, isopropyl, tert-propyl, butyl, isobutyl or tert-butyl can be used as C1-4-alkyl. Expedient representatives of the aromatic nitriles of the general formula I or II are 2-, 3- or 4-cyanopyridine, benzonitrile, fluoro-, chloro- or bromobenzonitrile, such as, for example, o-, m- or p-chlorobenzonitrile or 2-chloro-3-cyanopyridine. 3-Cyanopyridine is preferably used as aromatic nitrile having 4 to 10 carbon atoms.
The biotransformation is expediently carried out with addition of substrate in one portion or continuously such that the substrate concentration does not exceed 40% by weight, preferably 30% by weight.
The process is expediently carried out with resting (non-growing) cells.
Suitable media for the biotransformation are those customary in the specialist field, such as, for example, low molecular weight phosphate buffers, HEPES buffers, citrate buffers, borate buffers, the media according to Tables 1 to 3 or modified forms thereof such as, for example, those described in Example 8 (1) or TRIS/HCl buffers.
The biotransformation is expediently carried out at a temperature from 0 to 50xc2x0 C. and at a pH between pH 4.5 and pH 10, preferably at a temperature from 20 to 40xc2x0 C. and at a pH between pH 4.5 and pH 10.0.
In a particularly preferred embodiment, the biotransformation can be carried out in the presence of C1-4-alcohols. C1-4-alcohols employed can be methanol, ethanol, propanol or butanol. Methanol is preferably used.
After the reaction, the corresponding amides can then be isolated by customary working-up methods such as, for example, by crystallization.